Serum albumin, a protein of multiple functions and manifold applications, is one of the most extensively studied proteins in biochemistry. Over 25,000 literature citations involving the biochemistry and/or applications of serum albumins have been published since 1969. The mammalian serum albumins proteins are known to be the product of three tandem gene duplications, and possess high helical content (60%) and high cystiene content (17 disulphides) with approximate molecular weights in the range of 65,000 daltons. Complete amino acid sequences are known for bovine, rat, and human serum albumins. Although the principal function of serum albumin remains unknown, it contributes to many transport and regulatory processes. Many studies have focused on the multifunctional binding properties of this interesting protein which range from various metals, e.g. Ca and Cu, to fatty acids, hormones, and a wide spectrum of therapeutic drugs. The majority of these binding studies have involved the human serum albumin (HSA) and many have shown that the distribution, free concentration, and metabolism of various pharmaceuticals can be significantly altered as a function of the magnitude of binding to HSA.
A detailed knowledge of the three-dimensional structure of serum albumin is imperative in order to fully understand the binding modes as well as many of the physical properties of this multifaceted protein. In addition, since many newly developed pharmaceuticals are rendered less effective by HSA; it is apparent that the crystal structure of a serum albumin, particularly the human form, will find very broad and significant application in the area of rational drug design. Consequently, the serum albumins have been the subject of ongoing crystallographic investigation which includes the documentation of several crystal forms (Table 1). Because of difficulties with crystal size, quality, and/or reproducibility, the three-dimensional structure of a serum albumin remains unknown.
This invention is concerned with the methodology required to produce a new crystal form of HSA which can be grown reproducibly as large, relatively high quality crystals suitable for x-ray structure determination. Once the three dimensional structure has been determined it will become possible to learn the molecular details involved in the binding of the albumin with a large number of pharmaceutical compounds. This may be done by soaking crystals in an appropriate stabilizing solution which contains the drug molcules of interest. If the binding sites are available in this crystal form, a crystalline array containing the serum albumin protein and the drug molecule will be produced. Details of the molecular interaction between the drug and protein can then be determined by established procedures in x-ray crystallography.
Due to the multiple binding capabilities of HSA, knowledge of its three dimensional structure combined with suitable crystals, may also provide assistance in determining the structures of various small molecules and perhaps small proteins which have proven difficult to crystallize.
Crystals of human serum albumin have been known for some time. As early as 1952, large crystals of HSA had been grown. Detailed x-ray examination of these and other reported crystal forms, including crystals of Horse serum albumin were published by McClure and Craven in 1974 (1) See Table 1, below. Crystals of HSA have also been grown by Rao and co-workers (2). Table 1 summarizes the crystallographic data published to date on several human serum albumin crystal forms. According to Peters in a recent review (1985) on serum albumins (3):
Although readily crystallized, albumin has relinquished few of its secrets through x-ray crystallography to date. . . . Structural information from these crystals is awaited eagerly, but obtaining it appears to be fraught with obstacles. Low described monoclinic crystals as soft waxy, and crystals studied by Rao, et al. (1976) have tended to dissolve under study.
TABLE 1 __________________________________________________________________________ CRYSTAL DATA ON THE POLYMORPHS OF HUMAN SERUM ALBUMIN Crystal System Monoclinic Orthorhombic Orthorhombic Tetragonal Tetragonal __________________________________________________________________________ Space Group C2 P2.sub.1 2.sub.1 2.sub.1 P2.sub.1 2.sub.1 2 P4.sub.1 2.sub.1 2 P42.sub.1 2 or P4.sub.3 2.sub.1 2 Unit Cell a = 126.5(3) a = 155(1) a = 137.3(1) a = 84.0(5) a = 187(1) Dimensions b = 39.2(1) b = 83(1) b = 275(3) c = 276(3) c = 81(1) c = 135.2(3) c = 122(1) c = 58.02(2) B = 93.3(1) Unit Cell 668,900 1,570,000 2,125,000 1,947,000 2,832,000 Volume A.sup.3 Molecules/ 1 2 3 1 2 Asymmetric Unit Diffraction 2.7 3.7 3.0 3.8 2.9 Limits A Matthews 2.52 2.95 2.66 3.66 2.66 Coefficient* Solvent Fraction 52% 59% 54% 67% 54% References (1) (1) (2) (1) This work __________________________________________________________________________ *Based on HSA MW 66458 References 1. R. J. McClure and B. M. Craven, J. Mol. Biol. (1974) 83, 551-555 2. Rao, S. N. et al. (1976) J. Biol. Chem., 251, 3191-3193 3. T. Peters, Advances in Protein Chemistry, Vol. 37, pg 161-243, (1985)
Crystals of the monoclinic form reported by McClure and Craven appear to be the highest quality; unfortunately, the crystals are small and difficult to reproduce. It is difficult to adequately compare the crystal quality of the remaining tetragonal crystal form with the tetragonal crystal form reported here, since the diffraction resolution reported for that crystal form was obtained with a conventional sealed tube source.
It is therefore an object of this invention to provide HSA in the form of crystals amenable to use in x-ray diffraction studies.
Another object is to provide HSA crystals having a size of at least 0.5 mm in two dimensions.
Yet another object is to provide HSA crystals in a form suitable for drug binding studies.
Still another object is to provide a method of preparing such crystals.